Protonated species are often postulated as key intermediates in enzyme-induced as well as simple chemical reactions. In this research proposal a simple method of some generality is utilized; which when applied to biological reactions, can be used to establish not only the protonated species, but also the specific site of protonation. Irreversibly alkylated, charged analogs of the suspected protonated intermediates are synthesized, and then tested for the desired reactivity. During the past year we have concentrated our efforts toward the elucidation of the mechanism of the coenzyme, biotin. We have synthesized and tested an "irreversibly acidified" model of N-carboxybiotin in which "protonation" (in the form of an ethyl group) has occurred at the ureido oxygen atom. Treatment of this model compound with a series of surrogates for the biological acceptor molecule (i.e. water, tert-butanol, hydroxide, tert-butoxide, triethylamine, morpholine, potassium cyanide, phenyl lithium) did not lead to the transfer of the carbon dioxide moiety, instead dealkylation was observed in each case. The mechanisms of these reactions have been elucidated. We have also continued our studies on our proposed intramolecular mechanism reported last year. In this hypothesis, biotin acts as a template for the two reactants. Treatment of a series of acyl substituted imidazolidones and imidazolidinethiones with ketenes (ketene, diphenylketene, dichloroketene, and bis(trifluoromethyl)ketene) did not lead to the transfer of the acyl group. Notably, however, in the case of disubstituted imidazolidinethiones an isosteric S yields O substitution reaction occurred at the thione position with bis(trifluoromethyl)ketene and diphenylketene. The generality of this unique reaction has been investigated. BIBLIOGRAPHIC REFERENCES: Harold Kohn, Barbara Kohn, Marie Louise Steenberg, and Joseph P. Buckley, Syntheses and Pharmacological Activity of Substituted Imidazolidinethiones and Thiomidazolines, J. Med. Chem. 1977 (January issue), in press. Harold Kohn and Raymond E. Davis, Syntheses and Chemistry of N-Acyl Substituted Dihydroimidazo(2,1-b)thiazolium Salts, J. Org. Chem., 1977 (February issue), in press.